This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Little is known about the assembly of basal bodies and centrioles. To understand more about the duplication process, the cell growth and division cycle can be synchronized by cycles of light and dark to examine the early steps in basal body duplication. In addition, we have two mutants that may be of particular interest. The first is a strain that has the amorphous ring that is present at the base of wild-type basal bodies, but the mutant basal bodies do not assemble microtubules. Thus, its characterization is important to look at early steps. The second is an RNA interference strain that has significant reduction in the mRNA for a luecine rich repeat protein;it makes too many basal bodies. This protein appears to act as a negative regulator of basal body/centriole duplication and is not defective in basal body segregation. Synchronized strains will be characterized using material that has been high pressure frozen and freeze-substituted and embedded in plastic to reconstruct the entire basal body complex in serial tomographic reconstructions. To date, 4 serial tomograms of mitotic cells have been imaged that reveal developing probasal bodies. These are characterized by rings of singlet microtubules, similar to what has been described in Paramecium. In addition, cryosections of frozen hydrated material will also be cut and cryotomograms will be imaged to get detailed information of duplication intermediates in a manner close to the native state.